Identification of Human Plasma Proteins as Major Clients forthe Extracellular Chaperone Clusterin*

Received for publication, October 26, 2009, and in revised form, December 2, 2009 Published, JBC Papers in Press, December 7, 2009, DOI 10.1074/jbc.M109.079566

Amy R. Wyatt1 and Mark R. Wilson2

From the School of Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522, Australia

Clusterin (CLU) is an extracellular chaperone that is likely toplay an important role in protein folding quality control. Thisstudy identified three deposition disease-associated proteins asmajor plasma clients for clusterin by studying CLU-client com-plexes formed in response to physiologically relevant stress(shear stress, �36 dynes/cm2 at 37 °C). Analysis of plasma sam-ples by size exclusion chromatography indicated that (i) relativeto control plasma, stressed plasma contained proportionallymore soluble protein species of high molecular weight, and (ii)high molecular weight species were far more abundant whenproteins purified by anti-CLU immunoaffinity chromatographyfrom stressed plasma were compared with those purified fromcontrol plasma. SDS-PAGEandWestern blot analyses indicatedthat a variety of proteins co-purified with CLU from bothstressed and control plasma; however, several proteins wereuniquely present or much more abundant when plasma wasstressed. These proteins were identified by mass spectrometryas ceruloplasmin, fibrinogen, and albumin. Immunodot blotanalysis of size exclusion chromatography fractionated plasmasuggested that CLU-client complexes generated in situ are verylarge and may reach >4 � 107 Da. Lastly, sandwich enzyme-linked immunosorbent assay detected complexes containingCLU and ceruloplasmin, fibrinogen, or albumin in stressed butnot control plasma. We have previously proposed that CLU-client complexes serve as vehicles to dispose of damaged mis-folded extracellular proteins in vivo via receptor-mediatedendocytosis. A better understanding of these mechanisms islikely to ultimately lead to the identification of new therapies forextracellular protein deposition disorders.

Processes to attain and maintain native protein conforma-tions are vital for organismal viability. Conditions such as ther-mal and oxidative stress may cause proteins to partially unfoldand aggregate, a process thought to underpin the pathology ofmany so-called protein deposition diseases, including Alzhei-mer disease, arthritis, type II diabetes, age-related maculardegeneration (ARMD),3 and atherosclerosis (1–5). Although

intracellular mechanisms to monitor and control the foldingstate of proteins arewell characterized, corresponding extracel-lular mechanisms have yet to be established. It has been pro-posed that clusterin (CLU) is one of a small family of abundantextracellular chaperones that form part of a quality control sys-tem that acts to stabilize misfolded, aggregating extracellularproteins, and mediate their clearance from the body via recep-tor-mediated endocytosis and lysosomal degradation (6–8).CLU can stabilize proteins and prevent their precipitation

during exposure to a variety of stresses in vitro (9–14). Thisaction involves the formation of soluble high molecular weight(HMW) complexes incorporating both CLU and the stressedclient protein at an approximate mass ratio of 1:2 (CLU:client);when generated in vitro, these complexes have diameters of50–100 nm (14). CLU is found associated with extracellularprotein deposits in many serious diseases including drusen inARMD (15), renal immunoglobulin deposits in kidney disease(16), prion deposits in Creutzfeldt-Jakob disease (17), amyloidplaques in Alzheimer disease (18, 19), and also atheroscleroticplaques (20). The presence of CLU in these pathological depos-its suggests that it associates with unfolding proteins in vivo.Pathological protein depositionmay thus result when the chap-erone capacity of CLU and other machinery acting to preventthe accumulation of protein aggregates is exceeded by abnor-mally high levels of protein unfolding.In addition to misfolded client proteins, CLU also binds to

many native ligands; the nature of these latter interactionsremains largely uncharacterized; however, it is believed thatCLU has discrete binding sites for native ligands andmisfoldedclient proteins (12, 21). Typically, investigations of the chaper-one activity of CLU have been carried out usingmodel proteinsthat can be induced to unfold at experimentally convenientrates. It was previously shown that depletion of CLU fromhuman plasma increased the extent of plasma protein precipi-tation at both 60 (11) and 37 °C (22). However, the identity ofthe major chaperone client proteins for CLU in human plasmawas previously unknown. The identity of these client proteinsmay provide important insights into mechanisms underlyingthe development of extracellular protein deposition diseases. Inthis studywe exposedhumanplasma to physiologically relevantstress (gentle rotation to produce shear stress �36 dynes/cm2 at37 °C for 10 days) and used electrophoresis, Western blotting,and mass spectrometry to identify proteins that co-purifiedwith CLU from stressed (but not control) plasma by immuno-affinity chromatography. Immunodot blot assays of fractionsfrom size exclusion chromatography (SEC) and sandwichELISA were used to confirm that these putative endogenous

* This work was supported by Australian Research Council Grant DP0773555.1 Recipient of an Australian Postgraduate Award and an Australian Institute

for Nuclear Science and Engineering Postgraduate Award.2 To whom correspondence should be addressed: School of Biological Sci-

ences, University of Wollongong, Northfields Ave., Wollongong, Australia2522. Tel.: 61-2-4221-4534; Fax: 61-2-4221-4135; E-mail: mrw@uow.edu.

3 The abbreviations used are: ARMD, age-related macular degeneration; Az,azide; CERU, ceruloplasmin; CLU, clusterin; FGN, fibrinogen; HMW, high molec-ular weight; HSA, human serum albumin; PBS, phosphate-buffered saline; SEC,size exclusion chromatography; ELISA, enzyme-linked immunosorbent assay;HRP, horseradish peroxidase; GdnHCl, guanadine hydrochloride.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 6, pp. 3532–3539, February 5, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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plasma client proteins formed soluble HMW complexes withCLU in stressed human plasma.

EXPERIMENTAL PROCEDURES

Materials—All of the buffer salts were obtained from AjaxChemical Co. ortho-Phenylenediamine, 2-�-mercaptoethanol,BCA reagent, goat anti-FGN antiserum, goat anti-CERU anti-serum, rabbit anti-HSA antiserum, control goat serum, controlrabbit serum, and mouse anti-goat Ig-HRP were from Sigma-Aldrich. Sheep anti-rabbit Ig-HRP and control mouse IgG1were obtained from Millipore. Purified mouse monoclonalanti-CLU IgG1 (G7) was as described in Ref. 23.Isolation of Plasma Proteins Co-purifying with CLU—Whole

blood supplementedwith 20�M sodiumcitratewas centrifugedat 1,020 � g for 30 min to pellet cells. The plasma was collectedand supplemented with CompleteTM protease inhibitor mix-ture (RocheApplied Science) and 0.1% (w/v) sodiumazide (Az).One 50-ml aliquot was immediately filtered through a GF/Cmicrofiber glass filter (Whatman) and passed at 0.5 ml/minover monoclonal anti-CLU immunoaffinity columns (with anapproximate total bed volumeof 20ml), as previously described(25). The columns were subsequently washed with several col-umn volumes of phosphate-buffered saline (PBS; 137mMNaCl,2.7mMKCl, 1.5mMKH2PO4, 8mMNa2HPO4, pH 7.4) contain-ing 0.1% (w/v) Az (i.e. PBS/Az) before the bound protein waseluted using 2 M GdnHCl in PBS, pH 7.4. A second 50-ml ali-quot of plasma (from the same batch) was “stressed” as follows:plasma was held in a 100-ml Schott bottle in a Bioline 472 incu-bator shaker (Edwards Instrument Co.) rotating at 200 rpm at37 °C for 10 days. This is estimated to correspond to an approx-imate shear stress of 36 dynes/cm2 (24). Subsequently, thissample was processed as above using the same immunoaffin-ity procedure. In some cases, where it was not possible to usefreshly isolated plasma as the control (i.e. in sandwich ELISAand measurements of turbidity where absorbance readingswere required to be obtained concurrently), control plasma(from the same batch) was left static at room temperature for10 days.Plasma Protein Precipitation Assays—Total plasma protein

precipitation was assessed by microprotein assay and by spec-trophotometry. For each plasma sample (control or stressed),three 200-�l aliquots of plasma were filtered using separate0.45-�m Ultrafree�-MC centrifugal filter devices (Millipore).The precipitate collected on the membranes was extensivelywashed with PBS. The membranes were then covered with 200�l of 6 M GdnHCl in PBS and incubated at 60 °C with shakingovernight. Parafilm “M” (Pechiney Plastic Packaging) was usedto seal the membrane cups and ensure that no liquid volumewas lost during heating. The solutions were diluted 1:50 in PBSbefore a BCA assay was performed (80). In addition, control orstressed plasma was diluted 1:2 in PBS/Az in a quartz cuvette,and the A360 nm was measured using a WPA Biowave S2100diode array spectrophotometer (Biochrom). Statistical signifi-cance was determined using one-way analysis of variance andTukey Honestly Significant Difference (HSD).Size Exclusion Chromatography—SEC of 500 �l of whole

plasma or anti-CLU immunoaffinity eluate was carried outusing a SuperoseTM 6 10/300 column (GE Healthcare) equili-

brated in PBS/Az at the recommended flow rate of between 0.3and 0.5 ml/min, and the A280 nm was continuously monitoredusing an AKTA fast protein liquid chromatography system (GEHealthcare). Mass standards were from a commercial HMWcalibration kit (GE Healthcare). All of the buffers and sampleswere filtered (0.45�m)before use. For immunodot blot analysisof whole plasma, 0.5-ml fractions were collected.SDS-PAGE and Identification of Proteins by Mass

Spectrometry—Proteins purified by anti-CLU immunoaffinitychromatography from plasma samples (40 mg of total protein/lane) were separated on 8–15% SDS-PAGE gels using aHoeferTM SE 250/260 SDS-PAGE system (GE Healthcare).Mass spectrometry was outsourced commercially and per-formed by the Australian Proteome Analysis Facility. SelectedCoomassie Blue-stained bands were excised from gels using ascalpel blade. Excised bands were treated in-gel with peptide:N-glycanase F followed by tryptic digestion for 16 h. Matrix-assisted laser desorption ionizationmass spectrometrywas per-formed with an Applied Biosystems 4800 Proteomics analyzer.The spectra were acquired in reflectronmode in themass range700–3500 Da. The instrument was then switched to tandemmass spectrometry (tandem time-of-flight) mode where pep-tides from the mass spectrometry scan were isolated and frag-mented and then reaccelerated to measure their masses andintensities. The spectra were then examined using the data basesearch program Mascot (Matrix Science Ltd). High Mowsescores (� 69) in the peptide data base search indicated a likelymatch (p � 0.05).Western Blot and Immunodot Blot Analyses—For Western

blots, following SDS-PAGE performed as described above(loading 10 �g of total protein into each lane), the gels weresubsequently equilibrated in transfer buffer (26 mM Tris, 192mM glycine, 20% (v/v) methanol, pH 8.3), and the separatedproteins were transferred to nitrocellulose membrane using aMini Trans-Blot Cell Western blotting apparatus (Bio-Rad) at100 V for 1 h at 4 °C. The membrane was subsequently blockedovernight at 4 °C in 1% (w/v) heat-denatured casein in PBS.Primary and appropriate HRP-conjugated secondary antibod-ies diluted in heat-denatured casein in PBS following themanufacturer’s instructions were incubated in turn with themembrane for 1 h at 37 °C. The membrane was then washed in0.1% (v/v) Triton X-100 in PBS followed by PBS alone.Enhanced chemiluminescence detection was performed usingSupersignalWestern Pico substrate (Pierce) following theman-ufacturer’s protocols. Amersham Biosciences HyperfilmTM

ECL (GEHealthcare)was placed over themembrane in aKodakX-Omatic cassette to detect chemiluminescence. Once exposed,the film was removed from the cassette and developed usingKodak developer and fixer. Densitometry was performed using aGS 800 calibrated densitometer (Bio-Rad) andQuantityOne soft-ware (Bio-Rad).Theaverageoptical density/mm2of thebandswasused to estimate their relative quantities.For immunodot blots, plasma (control or stressed) was frac-

tionated over a SuperoseTM 6 10/300 column as describedabove. Four microliters of each 0.5-ml fraction was spottedonto nitrocellulose membrane and allowed to dry before a sec-ond 4-�l aliquot was applied. The dried membranes were thenblocked and processed as described above for Western blot.

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Sandwich ELISA—The wells of an ELISA plate (Greiner Bio-one)were coatedwith 10mg/ml purifiedG7 anti-CLUantibody(23) or control mouse IgG1 antibody and then blocked with 1%(w/v) bovine serum albumin in PBS or heat-denatured caseinin PBS. Control plasma (left static at room temperature) orstressed plasma was next added to the wells. Subsequently, pri-mary antisera reactive with either CERU, FGN, or HSA dilutedin the blocking solution (following the manufacturer’s instruc-tions) were added to the appropriate wells. Finally, an appropri-ate HRP-conjugated secondary antibody diluted in blockingsolution (following themanufacturer’s instructions) was added.All of the incubations were for 1 h at 37 °C with shaking, andextensive washing with PBS was performed between each step.The final wash was performed using 0.1% (v/v) Triton X-100in PBS followed by PBS alone. ortho-Phenylenediamine (2.5mg/ml) and 0.03% (v/v) H2O2 in 50 mM citric acid, 100 mM

Na2HPO4, pH 5, was then added to the wells of the plate. Thereaction was stopped using 1 M HCl before the A490 nm wasmeasured using a SpectraMax Plus384 microplate reader(Molecular Devices). Nonspecific binding was assessed usingspecies-matched anti-sera of irrelevant specificity and theappropriate secondary antibody. The results presented areadjusted for nonspecific binding by calculating the absorbancein wells coated with G7 relative to the absorbance in wellscoated with control mouse IgG1 (of irrelevant specificity) thatwere treated with the same plasma and primary and secondaryantibodies. Statistical significance was determined using Stu-dent’s t test.

RESULTS

Stress-induced Protein Precipitation—After 10 days, com-pared with control plasma held at room temperature, stressedplasmawas visiblymore turbid. The turbidity of control plasmaremained unchanged over this period; however, after 10 daysthe turbidity of stressed plasmawas significantly greater than atday 0 and that of control plasma at either time point (Fig. 1;F(3,8) � 433, Tukey HSD, p � 0.0001 in all cases). When per-formed as described, the BCA assay was unable to detect pro-tein in the filtrates of freshly obtained control plasma butdetected a mean � S.E. (n � 3) of 8.47 � 0.99 mg of protein inthe corresponding filtrates from 200-�l aliquots of stressedplasma.

Bias toward HMW Species as Detected by SEC of StressedPlasma and Anti-CLU Co-purifying Proteins—There were dif-ferences in the SEC profiles of freshly isolated control andstressed plasma samples (Fig. 2A). Most notably, the respectiveareas underneath the traces of A280 nm suggest that there wasless soluble protein in stressed plasma compared with an equalvolume of control plasma. Although there appeared to be lesssoluble protein in the stressed sample, the amount of proteineluting at the exclusion limit of the column (4 � 107 Da) wassimilar for both plasma samples (Fig. 2A), indicating that over-all a greater proportion of stressed plasma proteins migrated asvery large species. SECprofiles of proteins purified by anti-CLUimmunoaffinity chromatography from freshly isolated controlversus stressed plasmawere very different (Fig. 2B). TheA280 nmpeak eluting at the column exclusion limit was approximatelyfour times greater for proteins immunoaffinity-purified fromstressed plasma versus control plasma. Moreover, the majorityof species in the former sample were larger than �600 kDa,whereas those in the latter sample were predominately smallerin mass (Fig. 2B).Identification of Major Plasma Clients for Clusterin—When

equivalent amounts of total protein purified by anti-CLUimmunoaffinity chromatography from control and stressedplasma were analyzed side-by-side by reducing SDS-PAGE,many bands were detected in both samples (Fig. 3). A major

FIGURE 1. Turbidity of control and stressed plasma at day 0 and day 10.The plasma samples were diluted 1:2 in PBS on days 0 and 10, and the A360 nmwas measured. The figure shows the average A360 nm (n � 3 � standard error)for each sample. *, significantly increased turbidity relative to the three othersample types (Tukey HSD, p � 0.0001 in all cases). These results are represent-ative of three independent experiments.

FIGURE 2. SEC of stressed or control plasma (A) and anti-CLU immunoaf-finity-purified proteins (B) from stressed or control plasma. The samplesanalyzed were: whole plasma exposed to shear stress for 10 days or freshlyisolated control plasma (A) and proteins purified by anti-CLU immunoaffinitychromatography from the plasma samples described in A (B). The positions ofmolecular mass markers are indicated by labeled arrows; the exclusion limit(Vo) � 4 � 107 Da. The results shown are representative of two independentexperiments.

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band at �36 kDa, corresponding to the co-migrating � and �subunits of CLU, was detected in both samples; however, thiswas considerably more prominent in the control sample (Fig.3). In the sample prepared from control plasma, other proteinbands detected probably include those representing ApoA-I(�28 kDa) and IgG (light chain, �23 kDa; heavy chain, �50kDa), which are known to co-purify with CLU from normalhuman plasma (23, 25), as well as other minor contaminantsthat can be removed from CLU by subsequent ion exchangechromatography (26). The additional bands that were uniqueor more prominent in the sample prepared from stressedplasmawere also detected (Fig. 3). Several of these bands (Fig. 3,open arrows) were excised and subjected to mass spectrometryanalysis (multiple gels of various percentage acrylamide wereused to adequately resolve bands for mass spectrometry; how-ever, a single representative gel is shown). These analyses iden-tified CERU, FGN (� chain), and HSA as putative chaperoneclient proteins for CLU (Table 1).Western blot analysis was used to confirm that CERU, FGN,

and HSA co-purified with CLU from stressed plasma but not(or to a lesser extent) from control plasma (Fig. 4). Atmost, onlytraces of CERU and FGN were detected in the correspondingprotein fractions purified in the same way from control plasma(Fig. 4, A and B). There was measurable HSA in the fraction

prepared from control plasma; however, this was reproduciblysubstantially less than in the sample prepared from stressedplasma (Fig. 4C). A band representing HSA was detected atabout the position expected for the intact molecule (69 kDa,Fig. 4C), but the same was not true for CERU. Intact CERU hasamass of 122 kDa; however, it is prone to autolysis in plasma toyield fragments of 67, 53, and 20 kDa (27) that corresponded tothe approximate position of the major bands detected on theblot (Fig. 4A). Bands corresponding to the �, �, and � chains ofFGN (25, 56, and 48 kDa, respectively) were detected in the lanecontaining proteins from stressed plasma. In plasma, FGN ishighly susceptible to proteolysis and is present in many frag-mented and cross-linked forms (28, 29). Therefore, FGN bandsdetected between �29 and 36 kDa are likely to represent � and� chain degradation products (Fig. 4B).CLU formsHMWcomplexes with chaperone client proteins

in vitro (9, 10, 12, 14). If CLU-client protein complexes formedin situ in plasma are also large, then relative to unstressed con-trol plasma, a greater proportion of CLU and the client proteinswould be detected as HMW species in stressed plasma. Thiswas examined by using SEC to fractionate both control andstressed plasma on the basis of molecular size and then probingthe fractions with specific antibodies. Fig. 5 shows immunodotblot results for SEC fractions representing species corre-sponding to between 460 kDa and the exclusion limit of thecolumn (�4 � 107 Da) in mass probed with specific antibod-ies for CLU, CERU, FGN, and HSA. Strikingly, CLU and allthree client proteins were detected much more strongly inthe HMW fractions (�4 � 107 Da) prepared from stressedplasma compared with the corresponding fractions of con-trol plasma (Fig. 5). Most of the corresponding fractionsfrom control plasma showed little reactivity with antibodiesspecific for CLU, CERU, FGN, and HSA. This is consistentwith the known masses of these individual proteins (CLU, 61kDa; CERU, 122 kDa; FGN, 340 kDa; HAS, 69 kDa); the bulk

FIGURE 3. SDS-PAGE of proteins purified from stressed or control humanplasma by anti-CLU immunoaffinity chromatography. The proteins wereseparated using 12% SDS-PAGE under reducing conditions. The figure showsmolecular mass markers (left lane, masses indicated in kDa) and proteinsimmunoaffinity-purified from freshly isolated control plasma (labeled C) orstressed plasma (labeled S). The open arrows indicate bands that wereselected for mass spectrometry analysis. The solid black arrow indicates theposition of CLU. The results shown are representative of many independentexperiments.

FIGURE 4. Western blot analysis of proteins purified by anti-CLU immu-noaffinity chromatography from control or stressed human plasma. Theproteins separated by SDS-PAGE (10 �g of total protein loaded per lane)were transferred to nitrocellulose membrane and probed with anti-CERU(A), anti-FGN (B), or anti-HSA antisera (C). Each panel shows the position ofmolecular mass markers (left lanes, masses in kDa indicated), and proteinsimmunoaffinity-purified from freshly isolated control plasma (labeled C)or stressed plasma (labeled S). In B, the position of the �, �, and � subunitsof FGN are indicated. The results shown are representative of three inde-pendent experiments.

TABLE 1Identification of proteins by mass spectrometryProteins co-purifying with CLU from stressed plasma (indicated by open arrows inFig. 3) were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight. The spectra were examined using the data base search program Mascot(Matrix Science Ltd., London, UK). Mowse scores (�69) in the peptide data basesearch indicated a likely match (p � 0.05).

Band Mass Identity Mowse score Coverage Significance

kDa %1 17 CERU 175 35 p � 0.052 43 FGN (� chain) 157 54 p � 0.053 67 HSA and CERU 398 54 p � 0.05

97 34 p � 0.05

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of these proteins would elute in later fractions not repre-sented in this figure unless they were present as aggregates orcomplexes. The limited reactivity detected for the client pro-teins in the control plasma lanes probably represents lowlevel associations of these proteins with other unidentifiedplasma components or self-association (see “Discussion”).The results thus far indicated a preferential association of

CERU, FGN, and HSA with CLU in stressed human plasma. Ifthis association was the result of the chaperone action of CLU,then it would be expected that complexes incorporating bothCLU and one or more of these three putative client proteinswould be present in stressed plasma but not in control plasma.This was verified by sandwich ELISA. The wells of an ELISAplate were coated with an anti-CLU antibody (or an isotype-matched control antibody) and subsequently incubated withcontrol or stressed plasma. Bound CERU, FGN, or HSA werethen detected with specific antibodies. In this assay, absor-bance at 490 nm significantly above that of the controls willonly occur if species containing both CLU and a putativeclient protein are specifically bound to the anti-CLU anti-body on the wells. For all three putative client proteinstested, significantly greater absorbance was measured inwells incubated with stressed plasma compared with thoseincubated with control plasma (Fig. 6; CERU, FGN, and HSAt(4)� 9.06, t(4)� 4.85, and t(4)� 4.99, respectively; p � 0.02in all cases). When irrelevant control antibodies matched byisotype and species to the primary detection antibody wereused in the same assay, there was no significant differencebetween the wells incubated with stressed or control plasma(control antisera results; Fig. 6).

DISCUSSION

Exposure to physical and chemical stresses, such as elevatedtemperature, shear stress, oxidative stress, and UV irradiation,challenge all biological systems. One potential impact of thesestresses is damage to proteins, inducing misfolding, loss offunction, and aggregation. Although much is known aboutintracellular mechanisms that act to repair or dispose of stress-damaged proteins, little is known about corresponding extra-cellular mechanisms. When comparing the intracellular andextracellular environments, some stresses that can contributeto protein unfolding are greater in the latter. This includeshydrodynamic shear stress resulting from the hydraulic force ofplasma being pumped around the body (30, 31); normal arterialshear stress is between 10 and 70 dynes/cm2 (32). The extracel-lular environment is also more oxidizing than the cytosol (33).In this study, we used a constant temperature of 37 °C and anestimated shear stress (generated by orbital shaking) of �36dynes/cm2 (calculated using the formula of Ref. 24) to simulatephysiologically relevant extracellular conditions. Relative toplasma left stationary at room temperature, plasma stressed inthis way for 10 days showed increased turbidity and proteinprecipitation (Fig. 1). This result indicates that plasma proteins

FIGURE 5. Immunodot blot analyses of SEC-fractionated control orstressed human plasma. Freshly isolated control plasma (C) or stressedplasma (S) were fractionated using a SuperoseTM 6 column (V0 � 4 � 107 Da).Aliquots from each fraction were spotted onto a nitrocellulose membranethat was then incubated with antibodies against CLU (A), CERU (B), FGN (C), orHSA (D) followed by the appropriate HRP-conjugated secondary antibodyprior to development by ECL. The approximate molecular mass of the SECfractions is indicated at the top of the figure. The results shown are represent-ative of three independent experiments.

FIGURE 6. Detection of CLU-client protein complexes in stressed plasmaby sandwich ELISA. The results of sandwich ELISA detecting CLU-client pro-tein complexes containing CERU (A), FGN (B), or HSA (C) in human plasma. Theresults for “control antisera” were obtained using species-matched controlantisera as the primary detection antibody in each case. The results shown arethe average A490 nm (n � 3, � standard error) relative to the nonspecific bind-ing generated in wells coated with mouse IgG1 control antibody. *, increasedA490 nm relative to wells incubated with control plasma (stored static at 4 °C;Student’s t test, p � 0.02). The results shown are representative of three inde-pendent experiments.

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are prone to unfolding and aggregation under conditions oftemperature and shear stress that are likely to occur in vivo. Ifthis is the case, then intuitively, the human body must havesystems in place to control this problem.SEC indicated that the level of soluble protein remaining in

stressed plasma was less than in batch-matched control plasmaand that proportionately more proteins were present as HMWspecies in stressed plasma (Fig. 2). These findings may beexplained by the stress-induced partial unfolding of plasmaproteins, which subsequently aggregate to form increasinglylarge aggregates, some of which eventually become too large tostay in solution and form insoluble precipitate. Superimposedon this process, we suggest that under these conditions CLUforms large soluble complexes with probably many differentmisfolded plasma client proteins. This has the effect of amelio-rating the extent of protein precipitation measured. We havepreviously shown that the immunoaffinity depletion of CLUfrom human plasma significantly increased total protein pre-cipitation in this fluid when it was subsequently incubated ateither 60 °C (11) or 37 °C (22). Species �4 � 107 Da in stressedplasmamay be aggregates on their way toward becoming insol-uble or may be aggregates stabilized by extracellular chaper-ones such as CLU. CLU is itself physically very stable and doesnot aggregate or precipitate even in response to sustained heat-ing at 60 °C (9, 12–14). Thus the apparent depletion of CLUfrom the pool of soluble proteins in stressed plasma in the cur-rent study (Fig. 3) probably results from its incorporation intogrowing aggregates; under the conditions tested, CLU is unabletomaintain the solubility of all plasma proteins. Supporting theinvolvement of CLU in forming HMW chaperone-client com-plexes, SEC of proteins purified by anti-CLU immunoaffinitychromatography from stressed and control plasma indicatedthat, relative to the latter, the former was dominated by HMWprotein species. If it had been possible to elute proteins from theanti-CLU columns using nondenaturing conditions (instead ofthe 2 M GdnHCl used in this study), the difference in massprofile for the two samples might have been even greater. Ourrecent work showed that when CLU-client protein complexeswere generated from purified proteins in vitro, the mass ratiowas, for each of three different client proteins, �1:2, respec-tively (14). The complexes in the current study were purifiedfrom stressed human plasma by immunoaffinity chromatogra-phy, which (unavoidably) involved eluting bound complexeswith denaturing conditions. The harsh elution conditions arelikely to have led to partial disruption of the complexes, thusmaking measurements of stoichiometry rather meaningless.For this reason we did not measure the apparent stoichiometryof the complexes in the current study.SDS-PAGE analyses of proteins bound from control plasma

to anti-CLU columns were consistent with our experience inroutine purifications of plasma CLU (26). The protein bandsdetected (additional toCLU) are likely to represent knownCLUligands such as complement components (34), IgG (23) andApo-A1 (25). However, other bands were detected as uniquelypresent (or more abundant) in samples prepared from stressedplasma; these bands were regarded as corresponding to puta-tive endogenous plasma client proteins for the chaperoneaction of CLU. In this study, three bands were selected from

one-dimensional SDS-PAGE for further analysis. It isexpected that in the future, by applying a protein separationtechnique with greater resolution such as two-dimensionalSDS-PAGE, further putative client proteins will be identi-fied. Previous studies indicate that the chaperone action ofCLU is promiscuous (6, 9–14, 35); thus it is likely that CLUwill interact with most misfolding proteins, regardless oftheir identity, and that endogenous CLU-client complexeswill contain a heterogeneous mix of client proteins. How-ever, using the approaches described, identification of spe-cific plasma proteins as clients for CLU will depend on theirindividual relative abundances and stabilities. In the currentstudy, mass spectrometric analysis of the three bandsresolved by one-dimensional SDS-PAGE identified them ascorresponding to CERU, FGN, and HSA (Fig. 3 and Table 1).Western blot analyses showed that these three putative cli-ent proteins were preferentially detected in protein fractionsprepared by anti-CLU immunoaffinity chromatography ofstressed plasma versus control plasma (Fig. 4). The smallamounts of the putative client proteins co-purifying withCLU from control plasma (Fig. 4, control, C lanes) may bethe result of low levels of CLU-client complexes present infreshly isolated plasma. However, low level nonspecific bind-ing of client proteins to the anti-CLU immunoaffinity col-umns may also contribute in this regard, particularly in thecase of HSA, which is known to bind to many surfaces.Species much larger than expected for monomeric CLU,

CERU, FGN, and HSA were detected by immunodot blot anal-ysis of SEC-fractionated samples of both control and stressedplasma. CLU is well known to oligomerize in solution to form avariety of HMW aggregates (9, 14) and also to associate withHDL particles in plasma (25); this would account for the large(460 to �737 kDa) CLU-containing species detected in controlplasma (Fig. 5A). In the case of CERU, FGN, andHSA, the large(�460 kDa) species containing these proteins detected in con-trol plasma probably result from interactions between themand other plasma proteins.Many examples of such interactionsare known; for example, lactoferrin (36), protein C (37), andmyeloperoxidase (38) interact with CERU; at least 11 proteinsare known to interact with FGN including vitronectin (39), his-tidine-rich glycoprotein (40), and apolipoprotein(a) (41); andover 60 different proteins are believed to interact with HSA(42). Self-aggregation, which has been reported for both FGN(43) and HSA (44), may also account for the detection of theseproteins as larger species. Corresponding analyses of stressedplasma strongly detected CLU and each of the three client pro-teins CERU, FGN, andHSA in fractions representing still largerspecies approaching (or at) the exclusion limit of the SEC col-umn (4 � 107 Da; Fig. 5). This observation is consistent withCLU forming large HMW complexes with client proteins instressed plasma, as has been described for purified proteins inbuffered solutions (9, 10, 12, 14). Finally, using sandwichELISA,wewere able to confirm thatCLU-CERU,CLU-FGN, andCLU-HSAcomplexeswere present in stressed but not control plasma(Fig. 6). Collectively, these results indicate that under condi-tions ofmild, physiologically relevant stress, CLU forms solublechaperone-client complexes in plasma containing one or moreof CERU, FGN, and HSA.

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The observation that CLU is found associated with insolubleprotein aggregates in all protein deposition diseases in whichthis has been examined (45) suggests that CLU (i) binds tounfolding proteins in vivo and (ii) becomes incorporated intoprotein deposits when its chaperone action is overwhelmed byan excess of misfolded protein. All three CLU client proteinsidentified in this study are involved in protein deposition dis-eases. CERU, FGN, and HSA are found in ARMD depositsknown as drusen (15, 46–49). Additionally, plasma concentra-tions of CERU and FGN are higher in ARMD compared withnormal patients (50, 51). Co-localization of CLU with drusenproteins is very common in ARMD (15); furthermore, in thissame disease, non-drusen FGN deposition is implicated in theatrophy of the retinal pigment epithelium and choroidal neo-vascularization (52, 53). The progression of both atrophic andneovascular ARMD is supported by platelet activation, whichresults in secretion of growth factors and monocyte chemoat-tractants. Platelet activation is enhanced by unfolding of FGN(54) and results in the release of CLU from the platelets bydegranulation (55). The effects of the interaction between CLUand stressed FGNon platelet activation are currently unknown;this may have significance not only to ARMD but also to themany ischemic and atherosclerotic vascular conditions whereFGN deposition is known to occur (5, 56–58). As in ARMD,FGN deposition in such diseases is associated with the recruit-ment and activation of platelets and has been proposed as amechanism for vascular injury (59). Deposition of FGN hasbeen reported in breast cancer (60), mesothelioma (61), coloncancer (62), and lymphoma (63). Although the reasons for thisare unknown, one potential implication of this observation isthat FGN deposition promotes angiogenesis and cancer pro-gression. FGN deposits are also found in renal disease (64, 65),hereditary renal amyloidosis (66), and systemic lupus erythe-matosus (67). In Type 1 (insulin-dependent) diabetes mellitus,extracellular deposition ofHSA is observed around dermal cap-illaries (68), kidney (69, 70), skeletal muscle (71), and the thy-roid gland (72).Given the large number of diseases in which extracellular

protein deposition occurs (1–5, 8), characterization of mecha-nisms that clear damaged proteins in healthy individuals islikely to shed light on how protein deposition pathologies arise.In this study we have identified three plasma client proteinsthat bind to CLU during physiologically relevant stress. Theirdeposition in numerous conditions suggests that overwhelm-ing or disruption of normal activities that prevent their accu-mulation in healthy individuals is important in the progressionof disease. The in vivo interaction of CLU with these clientproteins (and others) in vivo is likely to be an important mech-anism to prevent the pathological deposition of misfoldedextracellular proteins. Significantly, it has been shown thatCLU knock-out mice develop progressive glomerulopathy,which is characterized by the accumulation of insoluble proteindeposits in the kidneys (73). This directly implicates CLU in theclearance of potentially pathological aggregating proteins,although the precise mechanism underlying this has yet to bedescribed. It has been proposed to occur via the receptor-me-diated endocytosis and subsequent lysosomal degradation ofextracellular chaperone-client protein complexes (6). Evidence

is rapidly accumulating that CLU and other abundant extracel-lular chaperones are key elements in a quality control systemfor extracellular protein folding (6–14, 21, 22, 35, 45, 74–79).This report is an important step toward a more completeunderstanding of the vitalmechanisms involved in extracellularprotein folding quality control.

Acknowledgment—Wollongong Hospital kindly donated humanblood for use in this study.

REFERENCES1. Carrell, R. W., and Lomas, D. A. (1997) Lancet 350, 134–1382. Carrell, R. W., and Gooptu, B. (1998) Curr. Opin. Struct. Biol. 8, 799–8093. Thomas, P. J., Qu, B. H., and Pedersen, P. L. (1995) Trends Biochem. Sci.

20, 456–4594. Soto, C. (2001) FEBS Lett. 498, 204–2075. Mullins, R. F., Russell, S. R., Anderson, D. H., and Hageman, G. S. (2000)

FASEB J. 14, 835–8466. Yerbury, J. J., Stewart, E.M.,Wyatt, A. R., andWilson,M. R. (2005) EMBO

Rep. 6, 1131–11367. Wilson, M. R., Yerbury, J. J., and Poon, S. (2008)Mol. BioSyst. 4, 42–528. Wyatt, A. R., Yerbury, J. J., Poon, S., and Wilson, M. R. (2009) Curr. Med.

Chem. 16, 2855–28669. Humphreys, D. T., Carver, J. A., Easterbrook-Smith, S. B., and Wilson,

M. R. (1999) J. Biol. Chem. 274, 6875–688110. Wilson, M. R., and Easterbrook-Smith, S. B. (2000) Trends Biochem. Sci.

25, 95–9811. Poon, S., Rybchyn, M. S., Easterbrook-Smith, S. B., Carver, J. A., Pan-

khurst, G. J., and Wilson, M. R. (2002) J. Biol. Chem. 277, 39532–3954012. Poon, S., Easterbrook-Smith, S. B., Rybchyn, M. S., Carver, J. A., andWil-

son, M. R. (2000) Biochemistry 39, 15953–1596013. Poon, S., Treweek, T. M., Wilson, M. R., Easterbrook-Smith, S. B., and

Carver, J. A. (2002) FEBS Lett. 513, 259–26614. Wyatt, A. R., Yerbury, J. J., and Wilson, M. R. (2009) J. Biol. Chem. 284,

21920–2192715. Crabb, J. W., Miyagi, M., Gu, X., Shadrach, K., West, K. A., Sakaguchi, H.,

Kamei, M., Hasan, A., Yan, L., Rayborn, M. E., Salomon, R. G., and Holly-field, J. G. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14682–14687

16. French, L. E., Tschopp, J., and Schifferli, J. A. (1992) Clin. Exp. Immunol.88, 389–393

17. Freixes,M., Puig, B., Rodríguez, A., Torrejon-Escribano, B., Blanco, R., andFerrer, I. (2004) Acta Neuropathol. 108, 295–301

18. Ghiso, J., Matsubara, E., Koudinov, A., Choi-Miura, N. H., Tomita, M.,Wisniewski, T., and Frangione, B. (1993) Biochem. J. 293, 27–30

19. Calero, M., Rostagno, A., Matsubara, E., Zlokovic, B., Frangione, B., andGhiso, J. (2000)Microsc. Res. Tech. 50, 305–315

20. Jordan-Starck, T. C., Lund, S. D., Witte, D. P., Aronow, B. J., Ley, C. A.,Stuart, W. D., Swertfeger, D. K., Clayton, L. R., Sells, S. F., Paigen, B., andHarmony, J. A. (1994) J. Lipid Res. 35, 194–210

21. Lakins, J. N., Poon, S., Easterbrook-Smith, S. B., Carver, J. A., Tenniswood,M. P., and Wilson, M. R. (2002) Biochemistry 41, 282–291

22. French, K., Yerbury, J. J., and Wilson, M. R. (2008) Biochemistry 47,1176–1185

23. Wilson,M. R., and Easterbrook-Smith, S. B. (1992)Biochim. Biophys. Acta1159, 319–326

24. Dardik, A., Chen, L., Frattini, J., Asada, H., Aziz, F., Kudo, F. A., andSumpio, B. E. (2005) J. Vasc. Surg. 41, 869–880

25. Jenne, D. E., Lowin, B., Peitsch, M. C., Bottcher, A., Schmitz, G., andTschopp, J. (1991) J. Biol. Chem. 266, 11030–11036

26. Park, D. C., Yeo, S.G.,Wilson,M. R., Yerbury, J. J., Kwong, J.,Welch,W. R.,Choi, Y. K., Birrer, M. J., Mok, S. C., andWong, K. K. (2008)Neoplasia 10,964–972

27. Kingston, I. B., Kingston, B. L., and Putnam, F.W. (1977) Proc. Natl. Acad.Sci. U.S.A. 74, 5377–5381

28. Connaghan, D. G., Francis, C. W., Lane, D. A., and Marder, V. J. (1985)

Identification of Chaperone Clients for Clusterin

3538 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 6 • FEBRUARY 5, 2010

by guest on March 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Blood 65, 589–59729. Gorinstein, S., Caspi, A., Goshev, I., Aksu, S., Salnikow, J., Scheler, C.,

Delgado-Licon, E., Rosen, A., Weisz, M., Libman, I., and Trakhtenberg, S.(2003) J. Agr. Food Chem. 51, 822–827

30. Ker, Y. C., and Chen, R. H. (1998) Lebenson Wiss Technol. 31, 107–11331. Schneider, S. W., Nuschele, S., Wixforth, A., Gorzelanny, C., Alexander-

Katz, A., Netz, R. R., and Schneider, M. F. (2007) Proc. Natl. Acad. Sci.U.S.A. 104, 7899–7903

32. Malek, A. M., Alper, S. L., and Izumo, S. (1999) JAMA 282, 2035–204233. Sitia, R., and Braakman, I. (2003) Nature 426, 891–89434. Tschopp, J., Chonn, A., Hertig, S., and French, L. E. (1993) J. Immunol.

151, 2159–216535. Yerbury, J. J., Poon, S., Meehan, S., Thompson, B., Kumita, J. R., Dobson,

C. M., and Wilson, M. R. (2007) FASEB J. 21, 2312–232236. Zakharova, E. T., Shavlovski, M. M., Bass, M. G., Gridasova, A. A., Pulina,

M. O., De Filippis, V., Beltramini, M., DiMuro, P., Salvato, B., Fontana, A.,Vasilyev, V. B., and Gaitskhoki, V. S. (2000) Arch. Biochem. Biophys. 374,222–228

37. Walker, F. J., and Fay, P. J. (1990) J. Biol. Chem. 265, 1834–183638. Segelmark, M., Persson, B., Hellmark, T., and Wieslander, J. (1997) Clin.

Exp. Immunol. 108, 167–17439. Podor, T. J., Campbell, S., Chindemi, P., Foulon, D. M., Farrell, D. H.,

Walton, P. D., Weitz, J. I., and Peterson, C. B. (2002) J. Biol. Chem. 277,7520–7528

40. Schinke, T., Koide, T., and Jahnen-Dechent, W. (1997) FEBS Lett. 412,559–562

41. Klose, R., Fresser, F., Kochl, S., Parson,W., Kapetanopoulos, A., Fruchart-Najib, J., Baier, G., and Utermann, G. (2000) J. Biol. Chem. 275,38206–38212

42. Zhou,M., Lucas, D. A., Chan, K. C., Issaq, H. J., Petricoin, E. F., 3rd, Liotta,L. A., Veenstra, T. D., and Conrads, T. P. (2004) Electrophoresis 25,1289–1298

43. Kim, S. H., Haimovich-Caspi, L., Omer, L., Yu, C. M., Talmon, Y., Wang,N. H., and Franses, E. I. (2007) Langmuir 23, 5657–5664

44. Atmeh, R. F., and Shabsoug, B. (1997) Electrophoresis 18, 2055–205845. Wilson, M. R., Yerbury, J., and Poon, S. (2008) inHeat Shock Proteins and

the Brain: Implications for Neurodegenerative Diseases and Neuroprotec-tion (Asea, A., and Brown, I., eds) Springer Science, Berlin, Germany

46. Sakaguchi, H., Miyagi, M., Shadrach, K. G., Rayborn, M. E., Crabb, J. W.,and Hollyfield, J. G. (2002) Exp. Eye Res. 74, 547–549

47. Umeda, S., Suzuki, M. T., Okamoto, H., Ono, F., Mizota, A., Terao, K.,Yoshikawa, Y., Tanaka, Y., and Iwata, T. (2005) FASEB J. 19, 1683–1685

48. Hageman, G. S., Luthert, P. J., Victor Chong, N. H., Johnson, L. V., Ander-son, D. H., and Mullins, R. F. (2001) Prog. Retin. Eye Res. 20, 705–732

49. Rodrigues, E. B. (2007) Ophthalmologica 221, 143–15250. Newsome, D. A., Swartz, M., Leone, N. C., Hewitt, A. T., Wolford, F., and

Miller, E. D. (1986) Invest. Ophthalmol. Vis. Sci. 27, 1675–168051. Smith,W.,Mitchell, P., Leeder, S. R., andWang, J. J. (1998)Arch. Ophthal-

mol. 116, 583–58752. Shiose, S., Hata, Y., Noda, Y., Sassa, Y., Takeda, A., Yoshikawa, H., Fuji-

sawa, K., Kubota, T., and Ishibashi, T. (2004) Graefes Arch. Clin. Exp.Ophthalmol. 242, 777–783

53. van der Schaft, T. L., Mooy, C. M., de Bruijn, W. C., and de Jong, P. T.(1993) Br. J. Ophthalmol. 77, 657–661

54. Barbucci, R., Lamponi, S., and Magnani, A. (2003) Biomacromolecules 4,

1506–151355. Witte, D. P., Aronow, B. J., Stauderman, M. L., Stuart, W. D., Clay, M. A.,

Gruppo, R. A., Jenkins, S. H., andHarmony, J. A. (1993)Am. J. Pathol. 143,763–773

56. Badimon, J. J., Lettino, M., Toschi, V., Fuster, V., Berrozpe, M., Chesebro,J. H., and Badimon, L. (1999) Circulation 99, 1780–1787

57. Mailhac, A., Badimon, J. J., Fallon, I. T., Fernandez-Ortiz, A., Meyer, B.,Chesebro, J. H., Fuster, V., and Badimon, L. (1994) Circulation 90,988–996

58. Ernst, E., and Resch, K. L. (1993) Ann. Intern. Med. 118, 956–96359. Massberg, S., Enders, G., Matos, F. C., Tomic, L. I., Leiderer, R., Eisen-

menger, S., Messmer, K., and Krombach, F. (1999) Blood 94, 3829–383860. Costantini, V., Zacharski, L. R., Memoli, V. A., Kudryk, B. J., Rousseau,

S. M., and Stump, D. C. (1991) Cancer Res. 51, 354–35861. Wojtukiewicz, M. Z., Zacharski, L. R., Memoli, V. A., Kisiel, W., Kudryk,

B. J., Rousseau, S. M., and Stump, D. C. (1989) Thromb Res. 55, 279–28462. Wojtukiewicz, M. Z., Zacharski, L. R., Memoli, V. A., Kisiel, W., Kudryk,

B. J., Rousseau, S. M., and Stump, D. C. (1989) J. Thromb. Haemost. 62,1062–1066

63. Costantini, V., Zacharski, L. R., Memoli, V. A., Kisiel, W., Kudryk, B. J.,Rousseau, S. M., and Stump, D. C. (1992) J. Lab. Clin. Med. 119, 124–131

64. Koffler, D., and Paronetto, F. (1965) J. Clin. Invest. 44, 1665–167165. Koffler, D., and Paronetto, F. (1966) Am. J. Pathol. 49, 383–39566. Uemichi, T., Liepnieks, J. J., and Benson, M. D. (1994) J. Clin. Invest. 93,

731–73667. Dujovne, I., Pollak, V. E., Pirani, C. L., andDillard,M. G. (1972)Kidney Int.

2, 33–5068. Chavers, B., Etzwiler, D., Barbosa, J., Bach, F. H., andMichael, A. F. (1984)

Diabetalogia 26, 415–41969. Westberg, N. G., and Michael, A. F. (1972) Diabetes 21, 163–16870. Miller, K., and Michael, A. F. (1976) Diabetes 25, 701–70871. Cohn, R.A.,Mauer, S.M., Barbosa, J., andMichael, A. F. (1978)Lab. Invest.

39, 13–1672. Raij, J., and Michael, A. F. (1981) The Lancet 1, 67173. Rosenberg, M. E., Girton, R., Finkel, D., Chmielewski, D., Barrie, A., 3rd,

Witte, D. P., Zhu, G., Bissler, J. J., Harmony, J. A., and Aronow, B. J. (2002)Mol. Cell. Biol. 22, 1893–1902

74. Hatters, D. M.,Wilson, M. R., Easterbrook-Smith, S. B., and Howlett, G. J.(2002) Eur. J. Biochem. 269, 2789–2794

75. Carver, J. A., Rekas, A., Thorn, D. C., andWilson,M. R. (2003) IUBMBLife55, 661–668

76. Yerbury, J. J., Rybchyn,M. S., Easterbrook-Smith, S. B., Henriques, C., andWilson, M. R. (2005) Biochemistry 44, 10914–10925

77. Stewart, E.M., Aquilina, J. A., Easterbrook-Smith, S. B., Murphy-Durland,D., Jacobsen, C., Moestrup, S., and Wilson, M. R. (2007) Biochemistry 46,1412–1422

78. Kumita, J. R., Poon, S., Caddy, G. L., Hagan, C. L., Dumoulin, M., Yerbury,J. J., Stewart, E. M., Robinson, C. V., Wilson, M. R., and Dobson, C. M.(2007) J. Mol. Biol. 369, 157–167

79. Yerbury, J. J., Kumita, J. R., Meehan, S., Dobson, C. M., andWilson, M. R.(2009) J. Biol. Chem. 284, 4246–4254

80. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H.,Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk,D. C. (1985) Anal Biochem. 150, 76–85

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Amy R. Wyatt and Mark R. WilsonChaperone Clusterin

Identification of Human Plasma Proteins as Major Clients for the Extracellular

doi: 10.1074/jbc.M109.079566 originally published online December 7, 20092010, 285:3532-3539.J. Biol. Chem. 

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